Genotoxicity testing of bovine lymphocytes exposed to epoxiconazole using alkaline and neutral comet assay

Abstract

Epoxiconazole belongs in the class of azoles which have been developed to protect crops from fungal diseases. The mechanism of action of these fungicides is to inhibit the specific cytochrome P450 enzyme (CYP), CYP51 (lanosterol 14?-demethylase) which contributes to ergosterol biosynthesis. Since ruminants and cattle are exposed to contaminants during grazing, they are a suitable experimental model for genotoxicity testing. In our experiment, epoxiconazole (EPX) (active agent, 99% of purity) was tested in vitro for its potential genotoxic and cytotoxic effects on bovine lymphocytes, isolated from whole peripheral blood. We exposed the lymphocytes to EPX at concentrations of 2.5, 5, 10, 25, 50 and 100 ?g/mL by two different ways: immediately after isolation of lymphocytes during 2 h in RPMI 1640 medium (without phytohaemagglutinin, PHA) as well as on the last 2 h of 48-h culture (with PHA). In a second case, we chose 48 h culture because the lymphocytes usually start DNA replication 24 h after the start of the cultures; therefore, we incubated the cells longer to obtain dividing (proliferating) cells. The levels of DNA damage were measured using alkaline and neutral comet assays. The results of alkaline comet assay showed the significantly increased percentage of DNA breaks in both lymphocytes in medium without PHA (2 h of exposure; non-proliferating cells) and lymphocytes cultured during 48 h in medium with PHA (exposure for the last 2 h of cultivation; proliferating cells). Similarly, neutral comet assay showed dose-dependent elevation of the DNA migration induced in both non-proliferating and proliferating lymphocytes treated with EPX when compared with negative controls. Our results suggest that epoxiconazole fungicide is capable of causing damage to the genetic material of the bovine cells.